A COMBINED WORKFLOW FOR IN-DEPTH CHARACTERIZATION OF CYSTEINE-CONJUGATED ANTIBODY DRUG CONJUGATES

Significance of the Topic

Antibody–drug conjugates (ADCs) combine the selectivity of monoclonal antibodies with potent cytotoxic drugs, but their inherent heterogeneity—variability in drug-to-antibody ratio (DAR), conjugation sites and distribution—poses significant analytical challenges. A robust, multidimensional characterization workflow is vital to ensure product quality, efficacy and regulatory compliance.

Objectives and Study Overview

The goal of this study is to develop an integrated analytical strategy for comprehensive profiling of cysteine-conjugated ADCs. Key objectives include:

• Determining DAR and drug distribution profiles at the intact protein level.
• Resolving and identifying positional isomers of conjugation sites.
• Profiling subunit fragments to assess conjugation patterns after enzymatic digestion.
• Mapping peptide-level conjugation sites and calculating occupancy ratios.

Methodology and Instrumentation

The workflow is organized into three analytical tiers:

• Intact Protein Analysis: HIC-UV and native SEC-LC/MS to automatically quantify DAR and drug distribution using integrated informatics.
• 2D LC/MS (HIC/RPLC): Heart-cutting HIC coupled to reversed-phase LC/MS to separate and assign positional isomers based on retention times and deconvoluted masses.
• Subunit Profiling: Enzymatic cleavage into heavy chain (HC), light chain (LC), Fab and Fc fragments, followed by RPLC-UV/MS to compare fragment isoforms.
• Peptide Mapping: Tryptic digestion and LC/MSE to identify conjugation sites and determine site-specific occupancy ratios.

Instrument details:

• LC: Waters ACQUITY H-Class Bio system (with 2D technology for HIC/RPLC).
• MS: Waters Xevo G2-S and G2-XS QTof platforms.
• Columns: Protein Pak Hi Res HIC, ACQUITY UPLC Protein BEH C4, ACQUITY UPLC Protein BEH SEC, CSH C18 for peptide separations.
• MS settings: 3 kV capillary voltage, 120–150 V cone voltage, source temperature 120–500 °C, desolvation at 250–350 °C, gas flow 600–800 L/h.

Main Results and Discussion

Intact Analysis: HIC-UV and SEC-LC/MS produced consistent DAR values across low, moderate and high drug loads, confirming method robustness. Native mass spectra after deglycosylation matched theoretical masses.
2D LC/MS: Heart-cut fractions from DAR 4 and DAR 6 samples yielded up to three distinct chromatographic peaks per fraction. Mass deconvolution allowed unambiguous assignment of positional isomers to specific cysteine residues.
Subunit Profiling: RPLC-UV overlays of reduced Fab/Fc fragments revealed seven subunit isoforms, corresponding to various conjugation patterns on HC and LC fragments.
Peptide Mapping: LC/MSE analysis identified 13 conjugated cysteine-containing peptides. Occupancy ratios were calculated from MS intensity, enabling precise quantification of site-specific modification (e.g., peptides with zero, one or two drug attachments).

Benefits and Practical Applications

This combined workflow offers:

• Automated and streamlined DAR determination, reducing manual intervention.
• Orthogonal confirmation of distribution profiles, enhancing confidence in data.
• Resolution of positional isomers, critical for batch consistency and regulatory filings.
• Comprehensive coverage from intact protein to peptide level, supporting process development, QA/QC and comparability studies.

Future Trends and Opportunities

Advancements such as ion mobility spectrometry and ultra–high-resolution MS may further improve heterogeneity profiling. Integrating machine learning for data analysis could accelerate interpretation of complex datasets. The workflow can be adapted to novel conjugation chemistries and payload classes, ensuring continued relevance as ADC technologies evolve.

Conclusion

The presented multi-dimensional approach integrates HIC-UV, SEC-LC/MS, 2D LC/MS, subunit profiling and peptide mapping to deliver a thorough characterization of cysteine-conjugated ADCs. It provides accurate DAR measurements, unambiguous positional isomer identification and detailed site-specific occupancy data, thereby enhancing the quality assessment of ADC products.

Reference

1. Details on the HIC-UV, SEC-LC/MS and RP-LC/MS analysis: 61st ASMS conference, poster number TP236
2. Details on 2D LC/MS analysis: 61st ASMS conference, poster number T2265